PDE5 inhibitors, sildenafil and vardenafil, reverse multidrug resistance by inhibiting the efflux function of MRP7 (ABCC10) transporter

PDE5 inhibitors, sildenafil and vardenafil, reverse multidrugresistance by inhibiting the efflux function of MRP7 (ABCC10)transporter

Jun-Jiang Chen1,2, Yue-Li Sun1,3, Amit K Tiwari1, Zhi-Jie Xiao1, Kamlesh Sodani1, Dong-Hua Yang4, Saraubh G Vispute1, Wen-Qi Jiang3, Si-Dong Chen2,*, and Zhe-Sheng Chen1,*

1Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St.John's University, Jamaica, New York, United States of America2Guangdong Key Laboratory for Molecular Epidemiology, School of Public Health, GuangdongPharmaceutical University, Guangzhou, Guangdong, China3Department of Medical Oncology, Cancer Center, Sun Yat-Sen University, Guangzhou,Guangdong, China4Biosample Repository, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States ofAmerica

SummaryPhosphodiesterase type 5 (PDE5) inhibitors are widely used in the treatment of male erectiledysfunction and in improving the breathing efficiency in pulmonary hypertension. Recently,several groups have evaluated the ability of PDE5 inhibitors for their anticancer activities.Previously, we have shown that sildenafil, vardenafil and tadalafil could reverse P-glycoprotein(P-gp, ABCB1)-mediated multidrug resistance (MDR). In the present study, we determinedwhether these PDE5 inhibitors have the potential to reverse multidrug resistance protein 7 (MRP7,ABCC10)-mediated MDR. We found that sildenafil and vardenafil dose-dependently enhanced thesensitivity of MRP7-transfected HEK293 cells to paclitaxel, docetaxel and vinblastine, whiletadalafil had only minimal effect. Accumulation and efflux experiments demonstrated thatsildenafil and vardenafil increased the intracellular accumulation of [3H]-paclitaxel by inhibitingthe efflux of [3H]-paclitaxel in HEK/MRP7 cells. In addition, immunoblot andimmunofluorescence analyses indicated that no significant alterations of MRP7 protein expressionand localization in plasma membranes were found after treatment with sildenafil, vardenafil ortadalafil. These results demonstrated that sildenafil and vardenafil reverse MRP7-mediated MDRthrough inhibition of the drug efflux function of MRP7. Our findings indicated a potentially noveluse of PDE5 inhibitors as an adjuvant chemotherapeutic agent in clinical practice.

IntroductionMultidrug resistance (MDR) is a phenomenon of resistance to mechanically and structurallyunrelated drugs. MDR leads to the failure of cancer treatment by chemotherapeutic drugs

*Corresponding author: Department of Pharmaceutical Science, St. John's University, Jamaica, New York, [email protected], Phone: 1-718-990-1432, Fax: 1-718-990-1877. *Guangdong Key Laboratory for Molecular Epidemiology, Schoolof Public Health, Guangdong Pharmaceutical University, Guangzhou, Guangdong, 510006, China; [email protected], Phone:86-020-39352100, Fax: 86-020-39352086..

Disclosure The authors have no conflicts of interest or disclosure.

List of Supporting Information Figure S1 (Cytotoxicity of PDE5 inhibitors to HEK293-pcDNA3.1 and HEK/MRP7 cells)

NIH Public AccessAuthor ManuscriptCancer Sci. Author manuscript; available in PMC 2013 August 01.

Published in final edited form as:Cancer Sci. 2012 August ; 103(8): 1531–1537. doi:10.1111/j.1349-7006.2012.02328.x.

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which are being still used as one of the most effective treatment option for cancers[1]. Oneof the major mechanisms behind the simultaneous resistance is the efflux of different drugsmediated by ATP-binding cassette (ABC) transporters from cancer cells[2]. ABC transportersuperfamily is a group of transmembrane proteins which are grouped into seven subfamilies(A~G) based on genome sequence similarities[3]. The ABCB1 (P-gp/MDR1), ABCG2(BCRP/MXR) and ABCCs (MRPs, multidrug resistance proteins) have been reported as themajor players to be involved in mediating resistance to certain anticancer drugs. ABCB1was the first discovered human ABC drug transporter, which transports a wide variety ofhydrophobic compounds, including some of the most common anticancer drugs such astaxanes, anthracyclines, vinca alkaloids and tyrosine kinase inhibitors (TKIs)[4, 5]. TheABCG2 can also transport several anticancer drugs among others such as antifolates,anthracyclines and TKIs[5–7]. The nine MRP members (MRP1~MRP9) involved in MDRrepresents the major part of the 12 members of the C subfamily of the human ABCtransporters[8]. The MRP subfamily can transport organic anions and anticancer drugs suchas anthracyclines, epipodophyllotoxins, vinca alkaloids and taxanes[9]. The MRP7(ABCC10), a member of MRP subfamily, is similar in topology to those of MRPs, 1, 2, 3and 6, with two nucleotide-binding domains and three transmembrane domains[10, 11].MRP7 is able to confer resistance to several natural product chemotherapeutic drugsincluding taxanes and vinca alkaloids which are also substrates of P-gp[12]. MRP7 has beenreported to confer resistance to vinorelbine and paclitaxel in non-small cell lung cancer(NSCLC) cells[13, 14] and to vincristine in human salivary gland adenocarcinoma (SGA)cells[15]. Hopper-Borge et al. have confirmed the in vivo functions of MRP7 using Mrp7knock-out mouse model and their results suggest that Mrp7 could affect in vivo tissuesensitivity toward taxanes[16].

It was recognized that developing inhibitors to ABC transporters may block transporter-mediated drug efflux function and re-sensitize MDR cancer cells to anticancer drugs. In thepast three decades, numerous broad-spectrum or specific inhibitors of ABC transportershave been discovered and tested in in vitro and in vivo studies[17]. However, most of theABC transporter inhibitors that were used as chemosensitizers have not been successfullyused in clinical cancer chemotherapy because of either adverse effects or toxicpharmacokinetic issues[2]. Another strategy for the reversal agent development isdiscovering new functions of the drugs that are clinically approved. Recently, our groupreported that several TKIs and phosphodiesterase-5 (PDE5) inhibitors could reverse P-gp-mediated MDR[18–23]. Since MRP7 shares similar substrates and functions with P-gp, it ispossible that P-gp modulators might also overcome MRP7-mediated MDR. Indeed, most ofthe reported MRP7 inhibitors could inhibit the function of P-gp[24–26]. In the present study,we performed experiments using MRP7-transfected HEK293 cells to determine whetherPDE5 inhibitors such as sildenafil, vardenafil and tadalafil could modulate MRP7-mediatedMDR.

Materials and methodsMaterials

Sildenafil, vardenafil and tadalafil were purchased from Toronto Research Chemicals Inc.(Ontario, Canada). Cepharanthine was generously provided by Daiichi SankyoPharmaceutical Co. Ltd (Tokyo, Japan). Dulbecco's modified Eagle's medium (DMEM),fetal bovine serum (FBS), penicillin/streptomycin and trypsin 0.25% were products ofHyclone (Logan, UT). Monoclonal antibody 14C10 (against GAPDH) was acquired fromCell Signaling Technology, Inc. (Danvers, MA). Polyclonal antibody D-19 (against MRP7)was obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Alexa flour 488 donkeyanti-goat secondary antibody for immunocytochemistry was purchased from MolecularProbes (Eugene, OR). [3H]-paclitaxel (46.5 Ci/mmol) was purchased from Moravek

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Biochemicals Inc (Brea, CA). Paclitaxel, docetaxel, vinblastine, dimethyl sulfoxide(DMSO), 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and other chemicalswere purchased from Sigma Chemical (St. Louis, MO).

Cell lines and cell cultureThe MRP7 cDNA was generously provided by Dr. Gary Kruh (University of Illinois,Chicago, IL) and inserted into the pcDNA3.1 expression vector. The MRP7 expressionvector and parental plasmid were introduced into HEK293 cells by electroporation aspreviously described[11]. Individual colonies were selected in medium containing G418(1mg/ml) and cultured for further analysis. All the cell lines were grown as adherentmonolayers in flasks with Dulbecco's modified Eagle's medium (DMEM) supplementedwith 10% bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin in ahumidified incubator containing 5% CO2 at 37°C.

Cytotoxicity assayMTT colorimetric assay was performed to analyze the drug sensitivity as previouslydescribed[19]. HEK293-pcDNA3.1 and HEK/MRP7 cells were seeded into 96-well plate intriplicate at 6000 cells/well and incubated in DMEM supplemented with 10% bovine serumat 37°C for 24 h. For determining the toxicity of PDE5 inhibitors, various concentrations ofsildenafil, vardenafil and tadalafil diluted with medium were added in to wells, respectively.For reversal effect of PDE5 inhibitors on the sensitivity of anticancer drugs in MRP7-overexpressing cells, three different non-toxic concentrations of sildenafil, vardenafil andtadalafil (1.25, 2.5, and 5 μM) were added into plates 1 h prior to the addition of thesubstrates of MRP7 (paclitaxel, docetaxel and vinblastine). After drug incubation of 68 h, 20μl MTT solution (4 mg/ml) was added into each well. The plates were further incubated for4 h, then the medium was discarded, and 100 μl of dimethylsulfoxide (DMSO) was addedinto each well to dissolve the formazan crystals. The absorbance was determined at 570 nmby an OPSYS Microplate Reader from DYNEX Technologies, Inc. (Chantilly, VA). Thedegree of resistance was calculated by dividing the IC50 values (concentrations required toinhibit growth by 50%) for the HEK/MRP7 cells by those of the parental HEK293-pcDNA3.1 cells. The Bliss method was used to calculate the IC50 values according tosurvival curves.

[3H]-paclitaxel accumulation and effluxThe effect of PDE5 inhibitors on the intracellular accumulation of paclitaxel in HEK293-pcDNA3.1 and HEK/MRP7 cell was measured using [3H]-paclitaxel. HEK293-pcDNA3.1and HEK/MRP7 cells were trypsinized and four aliquots from each cell line were suspendedin the medium, aliquots were pre-incubated with medium-only (control), sildenafil,vardenafil or tadalafil (5 μM each) at 37°C for 2 h, then incubated with 0.1 μM [3H]-paclitaxel for another 2 h. For efflux study, the cells were treated the same as drugaccumulation study, and then washed three times with ice-cold PBS, suspended in freshmedium with or without PDE5 inhibitors. Aliquots were evenly collected at various timepoints (0, 30, 60, 120 min). Samples from both accumulation and efflux experiments werewashed by ice-cold PBS thrice and placed in scintillation fluid and radioactivity wasmeasured in a Packard TRI-CARB® 1900CA liquid scintillation analyzer from PackardInstrument Company, Inc. (Downers Grove, IL).

Preparation of total cell lysates and immunoblotting analysisTo determine the effect of PDE5 inhibitors on the expression of MRP7, HEK/MRP7 cellswere incubated with 5 μM sildenafil, vardenafil or tadalafil for different time periods (0, 24,48 and 72 h) then harvested and rinsed twice with cold PBS. The total cell lysates were

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collected with Radioimmunoprecipitation assay (RIPA) buffer (1 × PBS, 1% Nonidet P-40,0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml phenylmethylsulfonyl fluoride, 10 μg/mlaprotinin, 10 μg/ml leupeptin) for 30 min with occasional rocking followed bycentrifugation at 12,000 rpm at 4°C for 15 min. The protein concentration was determinedby bicinchoninic acid (BCA™)-based protein assay (Thermo Scientific, Rockford, IL).Equal amounts of total cell lysates (40 μg of protein) were resolved by 4–12% sodiumdodecyl sulfate polycrylamide gel electrophoresis (SDS-PAGE) and electrophoreticallytransferred onto PVDF membranes. After being incubated in blocking solution containing5% skim milk in TBST buffer (10 mM Tris-HCL, PH 8.0, 150 mM NaCl and 0.1% Tween20) at room temperature for 1 h, the membranes were immunoblotted overnight withprimary antibodies anti-MRP7 (1:200 dilution) and anti-GAPDH (1:1000 dilution) at 4°C.Subsequently, the membranes were washed three times for 15 min with TBST buffer andincubated at room temperature for 2 h with horseradish peroxide (HRP)-conjugatedsecondary antibody (1:2000 dilution). The protein-antibody complex was detected by theenhanced Phototope TM-HRP Detection Kit (Cell Signaling, USA) and exposed to Kodakmedical X-ray processor (Kodak, USA). The protein expression was quantified by ScionImage software (Scion Co., Frederick, MD).

Immunofluorescence analysisHEK/MRP7 cells (1 × 104) were seeded in 24 well plates and cultured overnight. Sildenafil,vardenafil or tadalafil at 5 μM were added into the wells and then cultured at 37°C for 72 hin a humidified incubator containing 5% CO2. Cells were washed with PBS and fixed with4% paraformaldehyde for 15 min at room temperature and then rinsed with PBS three times.Non-specific reaction was blocked with 1% BSA for 1 h at room temperature. A polyclonalantibody D19 against MRP7 (1:200) was added and incubated overnight. Then cells wereincubated with Alexa Flour® 488 donkey anti-goat IgG (1: 2000) for 1 h at roomtemperature. DAPI was used for nuclear staining. Immunofluorescent images were takenwith an inverted microscope (model IX70; Olympus, Center Valley, PA) with IX-FLAfluorescence and CCD camera.

Statistical AnalysisAll experiments were repeated at least three times and the differences were determined byusing the Student's t-test. The statistical significance was determined at P < 0.05.

ResultsEffects of PDE5 inhibitors on the sensitivity of anticancer drugs in the HEK293-pcDNA3.1and HEK/MRP7 cells

Prior to analyzing the reversal efficacy of PDE5 inhibitors (sildenafil, vardenafil or tadalafil)on reversal MDR, we first tested their cytotoxic effects in HEK293-pcDNA3.1 and HEK/MRP7 cell lines using the MTT assay. The results showed that the HEK/MRP7 cell lines donot confer significant resistance to three PDE5 inhibitors (supplemental Figure 1). Then weinvestigated the cytotoxicity of anti-cancer drugs (paclitaxel, docetaxel or vinblastine) aloneand in combination with a PDE5 inhibitor (sildenafil, vardenafil or tadalafil, Figure 1) at itsnon-toxic concentrations (1.25, 2.5 and 5 μM) in the HEK293-pcDNA3.1 and HEK/MRP7cells. As shown in Table 1 and Figure 2, HEK/MRP7 cells compared to parental HEK293-pcDNA3.1 cells, exhibited a significant resistance to various MRP7 substrates such aspaclitaxel, docetaxel and vinblastine, which is consistent with our previous reports[22].Sildenafil, vardenafil and tadalafil dose-dependently decreased the IC50 values of the abovementioned MRP7 substrates for HEK/MRP7 cells. However, tadalafil showed the leastreversal effect. Cepharanthine, the known MRP7 inhibitor, as a positive control at 2.5 μMcompletely reversed the resistance of HEK/MRP7 cells to paclitaxel, docetaxel and

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vinblastine. In contrast, sildenafil, vardenafil or tadalafil did not significantly reverse theresistance of HEK/MRP7 cells to cisplatin, a non-substrate of MRP7 (P > 0.05, Table 1,Figure 2). In the parental HEK293-pcDNA3.1, the IC50 values of paclitaxel, docetaxel andvinblastine in the presence or absence of sildenafil, vardenafil or tadalafil showed nosignificant difference (P > 0.05, Table 1, Figure 2).

PDE5 inhibitors increase the intracellular accumulation of [3H]-paclitaxel in the HEK/MRP7cells

To further confirm the effects of PDE5 inhibitors on the drug efflux function of MRP7, theintracellular accumulation of [3H]-paclitaxel study was performed. The intracellularconcentration of [3H]-paclitaxel in HEK/MRP7 cells was significantly lower (28.5%) thanthat in parental HEK293-pcDNA3.1 cells (100%, Figure 3). After the cells were incubatedwith either sildenafil, vardenafil or tadalafil at 5 μM for 2 h, the intracellular accumulationof [3H]-paclitaxel in HEK/MRP7 cells was significantly increased by 3.3-, 3.7- and 2.1-fold,respectively, when compared to 2.5 μM of cepharanthine as a positive control by 3.8-fold(Figure 3). Neither PDE5 inhibitors nor cepharanthine significantly affected the intracellularlevels of [3H]-paclitaxel in HEK293-pcDNA3.1 cells (Figure 3).

PDE5 inhibitors inhibit the efflux of [3H]-paclitaxel mediated by MRP7 in HEK/MRP7 cellsTo ascertain whether the increase in the intracellular [3H]-paclitaxel accumulation in thepresence of sildenafil, vardenafil or tadalafil was due to the inhibition of [3H]-paclitaxelefflux by MRP7, we designed a time course study to measure intracellular [3H]-paclitaxellevels in the presence of sildenafil, vardenafil or tadalafil. As shown in Figure 4, HEK/MRP7 cells significantly extruded a higher percentage of intracellular [3H]-paclitaxel thanthat in HEK293-pcDNA3.1 cells. However, in the presence of sildenafil, vardenafil ortadalafil at 5 μM, there was significant decrease in the efflux of intracellular [3H]-paclitaxelat different time periods (0, 30, 60, 120 min) from HEK/MRP7 cells, but not in the parentalHEK293-pcDNA3.1 cells. The intracellular accumulation of [3H]-paclitaxel at 0 min was setas 100% and at 30, 60 and 120 min, the percentages were 62.43%, 39.47% and 23.44%,respectively, of the accumulated [3H]-paclitaxel that remained in HEK/MRP7 cells in theabsence of PDE5 inhibitors. When HEK/MRP7 cells were incubated with sildenafil, thepercentage of the intracellular [3H]-paclitaxel at 30, 60 and 120 min increased significantlyto 90.62%, 82.14% and 62.35%, respectively (Figure 4A). Vardenafil increased significantlythe percentages of the intracellular [3H]-paclitaxel at 30, 60 and 120 min to 95.34%, 93.58%and 63.92%, respectively (Figure 4B). Meanwhile, tadalafil at 30, 60 and 120 min increasedsignificantly the percentage of [3H]-paclitaxel accumulation to 76.44%, 63.41% and46.48%, respectively (Figure 4C). Sildenafil and vardenafil were more potent than tadalafil,which is consistent with the results in colorimetric growth assay and [3H]-paclitaxelaccumulation experiments.

PDE5 inhibitors do not alter the expression of MRP7Reversal of MRP7-mediated MDR can be achieved by either altering MRP7 expression orinhibiting MRP7 function. To evaluate the effects of sildenafil, vardenafil or tadalafil onMRP7 expression, HEK/MRP7 cells were treated with sildenafil, vardenafil or tadalafil at 5μM for 0, 24, 48, 72 h and the MRP7 expression levels were examined by Western blotanalysis. The results shown in Figure 5A indicated that any of PDE5 inhibitors does notsignificantly alter the protein expression levels of MRP7 in HEK/MRP7 cells.

PDE5 inhibitors do not alter the localization of MRP7Presumably, the transporters could be down regulated, if they are translocated or dislodgedfrom plasma membrane to cytosolic region. To rule out this possibility, we performed an

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immunofluorescence assay to examine whether the location of MRP7 was altered after thetreatment with PDE5 inhibitor. As shown in Figure 5B, there was no alteration of MRP7protein localization in plasma membranes after the treatment with sildenafil, vardenafil ortadalafil at 5 μM for 72 h. The Western blotting (Figure 5A) and immunocytochemical(Figure 5B) experiments suggested that all three PDE5 inhibitors do not alter the expressionand/or localization of the MRP7 transporter in HEK/MRP7 cells.

DiscussionPreviously, we reported for the first time that three PDE5 inhibitors, sildenafil, vardenafiland tadalafil, could reverse P-gp-mediated MDR by directly inhibiting the transport functionof P-gp[22, 23]. Meanwhile, the efficacy of tadalafil as a reversal agent for P-gp was weakerthan that of sildenafil and vardenafil. Furthermore, our in vivo experiments showed thatsildenafil significantly enhanced the sensitivity of anticancer drug on P-gp-mediated MDRcancer xenograft model in nude mice (unpublished data). In this study, we examinedwhether sildenafil, vardenafil or tadalafil could reverse MRP7-mediated anticancer drugresistance. For this study we chose well established HEK293-pcDNA3.1 and HEK/MRP7transfected cell lines[25]. The expression of MRP7 in HEK/MRP7 cell line was detected andconfirmed by immunoblot analysis (data not shown).

The PDE5 inhibitors, sildenafil and vardenafil were able to completely reverse the MDRmediated by MRP7 as evidenced with cytotoxicity assay data (Table 1). Sildenafil andvardenafil potently sensitized MRP7-overexpressing cells to MRP7 substrates paclitaxel,docetaxel and vinblastine. However, sildenafil and vardenafil did not sensitize the cells tocisplatin (non substrate of MRP7) and had no significant effect on the drug sensitivity of theparental HEK293-pcDNA3.1 cells. Consistent with the cytotoxicity results, the drugaccumulation data indicated that sildenafil and vardenafil significantly enhanced theintracellular accumulation of paclitaxel in HEK/MRP7 cells. Since MRP7 is a drug effluxpump that contributes to decrease of intracellular paclitaxel concentrations, the time courseefflux study was performed to further confirm the accumulation results. Indeed, the effluxstudy showed the efflux of intracellular paclitaxel was significantly blocked by sildenafil orvardenafil in the HEK/MRP7 cell lines in compare to that without being treated with anyPDE5 inhibitors. Hence the accumulation and efflux data along with cytotoxicity resultsindicate that sildenafil and vardenafil are targeting to MRP7 transporter.

The reversal effect of MRP7-mediated MDR by sildenafil or vardenafil could be either dueto the inhibition of MRP7 transporter function or downregulation of the expression of MRP7transporter protein. The immunoblot and immunofluorescence analyses data rule out thesecond possibility, where no alterations in protein expression and localization of MRP7transporter from plasma membranes were seen in HEK/MRP7 cells in the presence ofsildenafil or vardenafil at 5 μM for up to 72 h. These findings further strengthen our resultsindicating that sildenafil and vardenafil inhibit MRP7 efflux function rather thandownregulate MRP7 expression.

Sildenafil, vardenafil and tadalafil are cGMP-specific PDE5 competitive inhibitors that canprevent cGMP degradation. They are widely used in the treatment of male erectiledysfunction and pulmonary hypertension. These drugs have similar ring structure and areable to foster accumulation of the cellular cGMP leading to increase the relaxation ofvascular smooth muscle[27]. Recently, several groups have evaluated the ability of PDE5inhibitors on anticancer activities. PDE5 inhibitors were reported to involve inantiproliferation and proapoptotic mechanism in multiple carcinomas[28]. Sarfati et al.found that vardenafil and sildenafil could induce caspase-dependent apoptosis of B-chroniclymphocytic leukemia cells in vitro[29]. Moreover, Das et al. reported that sildenafil could

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enhance doxorubicin-induced apoptosis and up-regulate caspase-3 and caspase-9 activitiesin prostate cancer cells[30]. PDE5 inhibitors also have been reported to increase the tumorcapillary permeability and improve delivery of anticancer agents to brain tumor in a ratmodel[31]. In addition, it has been reported that PDE5 inhibitors were used as modulators ofthe anticancer immune response and could reverse tumor-induced immunosuppression[32].More recently, we reported that sildenafil and vardenafil could enhance the anticancer drugsensitivity of cancer cells by reversing P-gp-mediated MDR[22, 23]. These findingsdemonstrate a potentially novel use of PDE5 inhibitors as an adjuvant to chemotherapy andimmune therapy.

However, another PDE5 inhibitor, tadalafil, only at 5 μM concentration can significantlysensitize MRP7-overexpressing cells to chemotherapeutic drugs, and its efficacy is weakerthan that of sildenafil and vardenafil. Nonetheless, tadalafil has a longer half-life andduration of action than sildenafil and vardenafil in clinic[33]. Currently, the explanations forthis difference may be due to their structural-activity relationship or their ability to inhibitother PDE enzymes. The molecular configuration of tadalafil departs entirely from that ofboth sildenafil and vardenafil (Figure 1), whereas sildenafil and vardenafil differ only inparticular by their nitrogen atoms in the heterocyclic ring system[34–36]. Furthermore,sildenafil and vardenafil inhibit PDE1 and PDE6 more significantly than tadalafil[37].Future studies are needed to investigate the interactions between PDE families and MRP7and/or P-gp.

In conclusion, our findings demonstrate for the first time that sildenafil and vardenafil areable to reverse MRP7-mediated MDR by directly blocking drug efflux function of MRP7without altering MRP7 protein expression and localization from the plasma membranes.Hence, these PDE5 inhibitors might be inhibitors for multiple efflux pumps mediatingMDR, such as P-gp and MRP7. Additionally, sildenafil and vardenafil are already in clinicaluse, which makes them ideal candidates to be considered as an adjuvant to anticancerchemotherapy, especially in MRP7 and/or P-gp-mediated MDR.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Dr. Gary D. Kruh (University of Illinois at Chicago, USA) for HEK293 cell line and the MRP7 cDNA.We thank Kakenshoyaku Co (Japan) for providing cepharanthine.

Grant support: This work was supported by fund from National Institutes of Health (No.1R15CA143701 to Z.S.C.).

Abbreviations

PDE5 phosphodiesterase type 5

MDR multidrug resistance

ABC ATP-binding cassette

MRP7 (ABCC10) multidrug resistance protein 7

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Figure 1.Chemical structure of sildenafil (A), vardenafil (B) and tadalafil (C).

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Figure 2. Sildenafil and vardenafil reverse MRP7-mediated drug resistance in HEK/MRP7 cellsPanels A, B, C, D: the survival curves of HEK/MRP7 cells in the presence or absence ofsildenafil, vardenafil or tadalafilat at 5 μM and the parental HEK293-pcDNA3.1 cell at thedifferent concentrations of paclitaxel, docetaxel, vinblatine and cisplatin, respectively. Cellsurvival was determined by MTT assay as described in “Materials and Methods”. Datapoints represent the means ± SD of triplicate determinations. Experiments were performed atleast three independent times.

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Figure 3. The effects of sildenafil, vardenafil and tadalafil on the accumulation of [3H]-paclitaxelin HEK293-pcDNA3.1 and HEK/MRP7 cellsThe intracellular accumulation of [3H]-paclitaxel was measured by scintillation countingafter cells were pre-incubated with or without sildenafil, vardenafil, tadalafil orcepharanthine for 2 h at 37°C and then incubated with 0.1 μM [3H]-paclitaxel for another 2h at 37°C. Data points represent the means ± SD of triplicate determinations. Experimentswere performed at least three independent times. ** P < 0.01, for values versus those in thecontrol group.

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Figure 4. The effects sildenafil (A), vardenafil (B) and tadalafil (C) on the efflux of [3H]-paclitaxel in HEK293-pcDNA3.1 and HEK/MRP7 cellsCells were pre-incubated with or without sildenafil, vardenafil or tadalafil for 2 h at 37°Cand further incubated with 0.1 μM [3H]-paclitaxel for another 2 h at 37°C. Cells were thenincubated in the fresh medium with or without the PDE5 inhibitors for different time periodsat 37°C. After that cells were collected and the intracellular levels of [3H]-paclitaxel weremeasured by scintillation counting. A time course versus percentage of intracellular [3H]-paclitaxel was plotted (0, 30, 60, and 120 min). Data points represent the means ± SD oftriplicate determinations. Experiments were performed at least three independent times.

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Figure 5. Immunoblot detection (A) and Immunofluorescence detection (B) of MRP7 in HEK/MRP7 cells following incubation with PDE5 inhibitorsCell lysates were prepared from HEK/MRP7 cells incubated with 5 μM sildenafil,vardenafil and tadalafil for different time periods (0, 24, 48, and 72 h). Immunoblotdetection of MRP7 was done using polyclonal anti-MRP7 antibody, GAPDH was used as aninternal control for equal loading. Equal amounts (40 μg of protein) of total cell lysates wereused for each sample. The localization of MRP7 by immunofluorescence was done onparaformaldehyde fixed cells using polyclonal antibody D19 against MRP7 (1:200) andAlexa Flour® 488 donkey anti-goat IgG (1:2000). Propidium iodide was used for nuclearcounterstaining. Results from a representative experiment are shown. Similar results wereobtained in two other trials.

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Table 1

The effects of PDE5 inhibitors on the sensitivity of HEK293-pcDNA3.1 and HEK/MRP7 cells to paclitaxel,docetaxel, vinblastine and cisplatin.

CompoundsIC50 ± SD

a (nM)

HEK 293-pcDNA-3.1 HEK/MRP7

Paclitaxel11.64 ± 1.33 (1.0)

b 107.18 ± 11.25 (9.21)

+Sildenafil 1.25 μM 10.69 ± 1.04 (0.92) 45.47 ± 3.78** (3.91)

+Sildenafil 2.5 μM 9.96 ± 0.92 (0.86) 28.35 ± 2.41** (2.44)

+Sildenafil 5 μM 9.38 ± 0.86 (0.81) 13.37 ± 2.07** (1.15)

+Vardenafil 1.25 μM 10.85 ± 1.27 (0.93) 34.85 ± 3.36** (2.99)

+Vardenafil 2.5 μM 9.63 ± 0.89 (0.83) 19.86 ± 2.61** (1.71)

+Vardenafil 5 μM 9.14 ± 1.01 (0.79) 12.39 ± 1.54** (1.06)

+Tadalafil 1.25 μM 12.43 ± 0.95 (1.07) 93.78 ± 6.23 (8.06)

+Tadalafil 2.5 μM 11.41 ± 1.22 (0.98) 81.25 ± 7.16* (6.98)

+Tadalafil 5 μM 10.84 ± 0.77 (0.93) 68.36 ± 5.83** (5.87)

+Cepharanthine 2.5 μM 8.97 ± 1.18 (0.77) 11.81 ± 0.82** (1.01)

Docetaxel 5.73 ± 0.65 (1.0) 64.81 ± 5.19 (11.31)

+Sildenafil 1.25 μM 5.35 ± 0.47 (0.93) 38.29 ± 3.75** (6.68)

+Sildenafil 2.5 μM 5.28 ± 0.41 (0.92) 22.37 ± 2.97** (3.90)

+Sildenafi 1.5 μM 4.74 ± 0.56 (0.83) 7.36 ± 0.83** (1.28)

+Vardenafil 1.25 μM 5.72 ± 0.38 (1.0) 31.85 ± 3.62** (5.56)

+Vardenafil 2.5 μM 4.88 ± 0.55 (0.85) 17.95 ± 2.57** (3.13)

+Vardenafil 5 μM 4.59 ± 0.39 (0.80) 6.84 ± 0.79** (1.19)

+Tadalafil 1.25 μM 6.26 ± 0.43 (1.09) 62.48 ± 5.03 (10.91)

+Tadalafil 2.5 μM 6.35 ± 0.59 (1.11) 55.21 ± 5.87 (9.64)

+Tadalafil 5 μM 5.86 ± 0.54 (1.02) 47.85 ± 3.98* (8.35)

+Cepharanthine 2.5 μM 4.07 ± 0.52* (0.71) 5.88 ± 0.41** (1.03)

Vinblastine 11.19 ± 1.23 (1.0) 56.31 ± 4.61 (5.03)

+Sildenafill 2.5 μM 10.96 ± 0.95 (0.98) 36.19 ± 2.48** (3.23)

+Sildenafil 2.5 μM 10.27 ± 0.82 (0.92) 25.92 ± 3.04** (2.32)

+Sildenafil 5 μM 9.69 ± 0.97 (0.87) 14.37 ± 1.19** (1.28)

+Vardenafil 1.25 μM 10.73 ± 1.11 (0.96) 33.72 ± 2.89** (3.01)

+Vardenafil 2.5 μM 10.32 ± 1.03 (0.92) 21.45 ± 1.94** (1.92)

+Vardenafil 5 μM 9.48 ± 0.98 (0.85) 12.39 ± 1.05** (1.11)

+Tadalafil 1.25 μM 11.84 ± 0.88 (1.06) 51.28 ± 4.85 (4.58)

+Tadalafil 2.5 μM 11.51 ± 1.19 (1.03) 47.95 ± 4.11 (4.29)

+Tadalafil 5 μM 10.66 ± 1.08 (0.95) 40.54 ± 3.92* (3.62)


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CompoundsIC50 ± SD

a (nM)

HEK 293-pcDNA-3.1 HEK/MRP7

+Cepharanthine 2.5 μM 8.92 ± 1.07 (0.80) 11.35 ± 0.93** (1.01)

Cisplatin 1574.26 ± 84.95 (1.0) 1552.22 ± 74.39 (0.99)

+Sildenafil 5 μM 1730.35 ± 63.89 (1.10) 1629.48 ± 81.76 (1.04)

+Vardenafil 5 μM 1681.45 ± 102.34(1.07) 1714.60 ± 71.43 (1.09)

+Tadalafil 5 μM 1746.87 ± 75.02 (1.11) 1757.12 ± 109.15 (1.12)

+Cepharanthine 2.5 μM 1677.38 ± 59.32 (1.07) 1635.30 ± 62.78 (1.04)

Data are means ± SD of at least three independent experiments performed in triplicate.

aIC50: concentration that inhibited cell survival by 50%.

bFold-resistance was determined by dividing the IC50 values of HEK/MRP7 cells by the IC50 values of HEK293-pcDNA3.1 cells in the absence

or presence of sildenafil, vardenafil, tadalafil or verapamil.

*represents P < 0.05,

**represents P < 0.01.


PDE5 inhibitors, sildenafil and vardenafil, reverse multidrug resistance by inhibiting the efflux function of MRP7 (ABCC10) transporter

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